Antibody and utilization of the same

ABSTRACT

The present invention relates to antibodies specifically reacting with partial peptides in the C-terminal region of polypeptides having amino acid sequences represented by SEQ ID NOS: 1 through 8 or derivatives thereof, a method of quantifying urotensin II using the antibodies, and pharmaceutical compositions comprising the antibodies (e.g., for central nerve diseases, mental disorders, circulatory diseases, heart diseases, renal diseases, urinary tract disorders, or the like).

TECHNICAL FIELD

The present invention relates to an antibody having a bindingspecificity to a partial peptide in the C-terminal region of apolypeptide having the amino acid sequence represented by SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7 or SEQ ID NO: 8, or a derivative of the polypeptide. Moreparticularly, the present invention relates to an antibody, which isuseful for developing a method of quantifying the aforesaid polypeptideor derivatives thereof, based on an antigen-antibody reaction, fordevelopment of diagnostic agents and preventive/therapeutic agents fordiseases associated with the polypeptide or derivatives thereof, etc.

BACKGROUND ART

It is known that various endogenous physiologically active peptides suchas angiotensin II, bradykinin, endothelin, etc. relatet a regulation ofcardiovascular systems such as cardiac function, blood pressure, etc. inmammals including human. In addition to these peptides, urotensin II wasnewly found to associate with cardiovascular systems recently, and hasdrawn attention as a new peptide of cardiovascular systems. Urotensin IIis a peptide originally found in fish urophysis and is known toparticipate in cardiovascular regulation, osmotic regulation, lipidmetabolism, etc. On the other hand, fish urotensin II was found to havehypotensive actions on mammals such as rat, etc. by intravenousadministration or vasoconstrictor or vasodilatation actions on vesselspecimens and the specific binding to labeled urotensin II was confirmedin the membrane preparations from rat vessels. It was thus predictedthat the homolog of urotensin II would be present also in mammals tofunction as an endogenous peptide and its specific receptor would bepresent (J. Exp. Zool., 275, 226-238, 1996). And predictably, it wasshown that the precursor gene of urotensin II was present in fish and afrog and further in mammals including mouse, rat and human (Proc. Natl.Acad. Sci. USA, 95, 15803-15808, 1998, FEBS Lett., 457, 28-32, 1999, WO01/04298). Furthermore, urotensin II as a mature peptide processed fromthe precursor gene was purified and isolated from porcine spinal cords,indicating that urotensin II was actually present as a peptide also inmammals (Biochem. Biophys. Res. Commun., 265, 123-129, 1999, WO00/32627). It was further clarified that human and rat GPR14 (SENR)(Genomics, 29, 335-344, 1995, Bichem. Biophys. Res. Commun., 209,752-759, 1995), which is an orphan receptor with unknown ligand, was afunctional receptor for urotensin II, based on the reactivity found inGPR14 receptor protein-expressed animal cells added with urotensin II asa ligand candidate (WO 01/04298, Nature, 401, 282-286, 1999, Biochem.Biophys. Res. Commun., 266, 174-178, 1999, Nature Cell Biol., 1,383-385, 1999), or by purifying urotensin II as a ligand activesubstance from animal tissue extracts using the reactivity of thereceptor-expressed cells as an indicator (Biochem. Biophys. Res.Commun., 265, 123-129, 1999, WO 00/3262).

Prior to the discovery of homologous peptides in mammals and theirreceptors, it had already been found that urotensin II had an extremelypotent vasoconstrictive action, using goby urotensin II and rat thoracicaorta (Am. J. Phys., 21, R361-R366, 1987, Eur. J. Pharmacol., 149,61-66, 1988), and the activity was confirmed also by using humanurotensin II (Nature, 401, 282-286, 1999). It was further demonstratedthat by intravenous administration in monkeys, urotensin II causedsystemic vasoconstriction to decrease blood flow and induced heartfailure by coronary vasoconstriction (Nature, 401, 282-286, 1999). Fromthe foregoing, it was predicted that urotensin II may be involved inonset of heart disease, etc. as a new peptide associated withcardiovascular systems. However, subsequent investigations usingisolated human vessels indicated that urotensin II did not always inducemarked vasoconstriction in human coronary vessels or small vessels andthe behavior on the circulatory system in human was not very potent (Br.J. Pharmacol., 131, 441-446, 2000, Am. J. Physiol. Heart Circ. Physiol.,280, H925-H928, 2001, Circulation, 103, 1378-1381, 2001). In theexperiments where urotensin II was administered to human, there are bothreports urotensin II reduced forearm blood flow (Br. J. Pharmacol., 135,25-27, 2002) and did not affect the blood flow (Cardiovasc. Res., 53,341-347, 2002). Recently, it was reported that the expression ofurotensin II and its receptor right ventricle was enhanced in rat whichdeveloped pulmonary hypertension and right ventricular hypertrophy underlow-oxygen condition (Heart Vessels, 16, 64-68, 2002) and in human aswell, the expression of urotensin II was enhanced in the myocardium ofpatients with congestive heart failure (Lancet, 359, 1990-1997, 2002).Moreover, since it is reported that urotensin II reportedly inducedhypertrophy in cultured cardiomyocytes of rat (FEBS Lett., 508, 57-60,2001), it is suggested the possibility that urotensin II mightparticipate in development of cardiac hypertrophy to cause heartfailure. In addition, it is reported that blood levels of urotensin IIIhave been found to be elevated in patients with heart failure (Lancet,360, 545-546, 2002). Furthermore, it was reported that blood or urinaryconcentrations of urotensin II were increased in patients with renaldysfunction, etc. (Lancet, 358, 810-811, 2001, J. Hypertension, 19,2185-2190, 2001), suggesting that urotensin II might take part in renalfunctions. It is also reported that GPR14 coexisted with cholinergicneurons in the mesopontine tegmental area (Brain Res., 923, 120-127,2001) and intracerebroventricular injection of urotensin II elicited anincrease in behavioral responses or increased anxiety in tests usingrats (Psychopharmacology, 155, 426-433, 2001, WO 02/14513), suggestingthat some central actions would be involved.

Further investigations are required for the involvement of urotensin IIin physiological effects or diseases and hence, it has been earnestlydesired to develop an assay system for detecting/quantifying urotensinII in a simple manner with high sensitivity.

DISCLOSURE OF INVENTION

The present inventors have made extensive investigations to solve theforegoing problems and as a result, produced a plurality of monoclonalantibodies capable of recognizing urotensin II and developed anexcellent method of determining urotensin II by using the antibodies.Further investigations have been made to accomplish the presentinvention.

That is, the present invention provides the following features, and thelike.

-   -   (1) An antibody reacting specifically reacting with a partial        peptide in the C-terminal region of a polypeptide having the        amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2,        SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID        NO: 8, or a derivative of said polypeptide.    -   (2) The antibody according to (1), which specifically reacts        with a partial peptide in the C-terminal region of a polypeptide        having the amino acid sequence represented by SEQ ID NO: 1, SEQ        ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:        6, or a derivative of said polypeptide.    -   (3) The antibody according to (1), which specifically reacts        with a peptide having the amino acid sequence represented by SEQ        ID NO: 9.    -   (4) The antibody according to (1), wherein the partial peptide        in the C-terminal region is a peptide having (i) the 5-10 amino        acid sequence in SEQ ID NO: 1, (ii) the 6-11 amino acid sequence        in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:        6, (iii) the 8-13 amino acid sequence in SEQ ID NO: 5, (iv) the        2-7 amino acid sequence in SEQ ID NO: 7, or (v) the 8-13 amino        acid sequence in SEQ ID NO: 8.    -   (5) The antibody according to (1), which is a monoclonal        antibody.    -   (6) The antibody according to (1), which is labeled.    -   (7) The antibody according to (1), which is a neutralizing        antibody.    -   (8) The antibody according to (7), which neutralizes the        activity of a polypeptide having the amino acid sequence        represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID        NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or a        derivative thereof.    -   (9) The antibody according to (7), which neutralizes the        activity of a polypeptide having the amino acid sequence        represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID        NO: 5 or SEQ ID NO: 6.    -   (10) The antibody according to (5), which is shown by        AUII5-6-10a capable of being produced from a hybridoma cell        shown by AUII5-6-10 (FERM BP-8221).    -   (11) The antibody according to (5), which is shown by        AUII103-5-41a capable of being produced from a hybridoma cell        shown by AUII103-5-41 (FERM BP-8220).    -   (12) A hybridoma cell capable of producing the antibody        according to (5).    -   (13) The hybridoma cell according to (12), which is shown by        AUII103-5-41 (FERM BP-8220).    -   (14) The hybridoma cell according to (12), which is shown by        AUII5-6-10 (FERM BP-8221).    -   (15) A method of producing the antibody according to (5), which        comprises culturing the hybridoma cell according to (12) in vivo        or in vitro and collecting the antibody according to (5) from        the body fluid or its culture.    -   (16) A pharmaceutical comprising the antibody according to (1).    -   (17) The pharmaceutical according to (16), which is a        preventive/therapeutic agent for central nerve diseases, mental        disorders, circulatory diseases, heart diseases, renal diseases        or urinary tract disorders.    -   (18) A diagnostic agent comprising the antibody according to        (1).    -   (19) The diagnostic agent according to (18), which is a        diagnostic agent for central nerve diseases, mental disorders,        circulatory diseases, heart diseases, renal diseases or urinary        tract disorders.    -   (19a) The diagnostic agent according to (18), which is a        diagnostic agent for mental disorders.    -   (20) A method of quantifying a polypeptide having the amino acid        sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:        3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8, or        a derivative thereof, which comprises using the antibody        according to (1).    -   (21) A method of quantifying a polypeptide having the amino acid        sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:        3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8, or        a derivative thereof, in a test fluid, which comprises        competitively reacting the antibody according to (I), a test        fluid and a labeled form of polypeptide having the amino acid        sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:        3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ        ID NO: 8, or a derivative of said polypeptide, and determining a        ratio of the labeled polypeptide having the amino acid sequence        represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID        NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8,        or a derivative thereof, bound to the antibody.    -   (22) A method for diagnosis of a disease associated with a        polypeptide having the amino acid sequence represented by SEQ ID        NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,        SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or a derivative        thereof, which comprises using the antibody according to (1).    -   (23) A method of preventing/treating central nerve diseases,        mental disorders, circulatory diseases, heart diseases, renal        diseases or urinary tract disorders, which comprises        administering an effective dose of the antibody according to (1)        to a mammal.    -   (24) Use of the antibody according to (1) for manufacturing a        preventive/therapeutic agent for central nerve diseases, mental        disorders, circulatory diseases, heart diseases, renal diseases        or urinary tract disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of antibody titers of mice immunized with gobyurotensin II, which were examined using biotinylated goby urotensin IIand HRP-labeled avidin. In the figure, symbols □ (open square), ▪(closed square), ◯ (open circle), ● (closed circle), Δ (open triangle),▴ (closed triangle), ⋄ (open diamond) and ♦ (closed diamond) designatemice No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7 and No. 8,respectively.

FIG. 2 shows typical examples for screening of hybridomas after cellfusion using mice immunized with goby urotensin II. In the figure,symbols □ (open square) and ▪ (closed square) designate the results withaddition of no porcine urotensin II-1 and the results with addition ofporcine urotensin II-1.

FIG. 3 shows the results of reactivities of AUII5-6-10a with humanurotensin II (-□-), porcine urotensin II-1 (-◯-), bovine urotensin II(-Δ-), rat urotensin II (-⋄-) and goby urotensin II (-x-), which wereexamined by competitive EIA using biotinylated goby urotensin II andHRP-labeled avidin.

FIG. 4 shows the results of reactivities of AUII103-5-41a with humanurotensin II (-□-), porcine urotensin II-1 (-◯-), bovine urotensin II(-Δ-), rat urotensin II (-⋄-) and goby urotensin II (-x-), which wereexamined by competitive EIA using biotinylated goby urotensin II andHRP-labeled avidin.

FIG. 5 shows the results of neutralizing effects of UII5-6-10a onarachidonate metabolite releasing activities of human urotensin II(-□-), porcine urotensin II-1 (-◯-), bovine urotensin II (-Δ-), raturotensin II (-⋄-) and goby urotensin II (-x-) from rat GPR14 receptorexpression CHO cells.

FIG. 6 shows the suppressing effects of AUII5-6-10a on anxiety-likebehaviors by intraventricular administration. In the figure, A, B and Con the abscissa designate the mouse IgG group (non-stressed), the mouseIgG group (exposed to restraint stress) and the AUII5-6-10a group(exposed to restraint stress), respectively, and the ordinate designatesthe count of peeping behavior.

BEST MODE FOR CARRYING OUT THE INVENTION

Throughout the specification, the proteins (polypeptides) arerepresented in accordance with the conventional way of describingpeptides, that is, the N-terminus (amino terminus) at the left hand andthe C-terminus (carboxyl terminus) at the right hand. In the proteinsused in the present invention, including a polypeptide having the aminoacid sequence represented by SEQ ID NO: 1, the C-terminus may be in anyform of a carboxyl group, a carboxylate, an amide and an ester.

The polypeptide having the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7 or SEQ ID NO: 8, and derivatives thereof are sometimescollectively referred to as the peptide of the present invention. Inaddition, the polypeptide having the amino acid sequence represented bySEQ ID NO: 9 and derivatives thereof are also included in the peptide ofthe present invention.

The derivatives described above include, for example, peptides wherein apart of amino acid residues in the amino acid sequence represented bySEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 are substituted with asubstitutable group, a part of the amino acid residues is deleted, theamino acid residues, etc. are added/inserted, and the like.

Examples of the derivatives of polypeptide having the amino acidsequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 include(i) those wherein at least 1 or 2 (e.g., 1 to 5, preferably 1 or 2)amino acids in the amino acid sequence described above are deleted, (ii)those, to which at least 1 or 2 (e.g., 1 to 5, preferably 1 or 2) aminoacids in the amino acid sequence described above are added, (iii) thosewherein at least 1 or 2 (e.g., 1 to 5, preferably 1 or 2) amino acids inthe amino acid sequence described above are inserted, or (iv) thosewherein at least 1 or 2 (e.g., 1 to 5, preferably 1 or 2) amino acids inthe amino acid sequence described above are substituted with other aminoacids.

The derivatives described above further include those, wherein a part ofamino acid residues in the polypeptide having the amino acid sequencerepresented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 is substitutedwith substitutable group(s) (e.g., Cys, hydroxyl group, etc.), thosewherein a part of the amino acid residues is deleted and a part of theamino acid residues is substituted with a substitutable group(s) (e.g.,Cys, hydroxyl group, etc.), and the like.

As the partial peptide in the C-terminal region of the peptide of thepresent invention, there are, for example, (i) a peptide having the 5-10amino acid sequence in SEQ ID NO: 1, (ii) a peptide having the 6-11amino acid sequence in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQID NO: 6, (iii) a peptide having the 8-13 amino acid sequence in SEQ IDNO: 5, (iv) a peptide having the 2-7 amino acid sequence in SEQ ID NO:7, (v) a peptide having the 8-13 amino acid sequence in SEQ ID NO: 8,(vi) peptides wherein a part of amino acid residues (e.g., one aminoacid residue) in these peptides is substituted with a substitutablegroup, and the like.

As the partial peptide in the N-terminal region of the peptide of thepresent invention, there are, for example, (i) a peptide having the 1-5amino acid sequence in SEQ ID NO: 1, (ii) a peptide having the 1-6 aminoacid sequence in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:6, (iii) a peptide having the 1-8 amino acid sequence in SEQ ID NO: 5,(iv) a peptide having the 1-8 amino acid sequence in SEQ ID NO: 8, (v)peptides wherein a part of amino acid residues (e.g., one amino acidresidue) in these peptides is substituted with a substitutable group,and the like.

The antibodies specifically reacting with the partial peptides at the Cterminus of the peptide of the present invention may be any antibodies,which are capable of specifically reacting with the partial peptides atthe C-terminus of the peptide of the present invention, and includeantibodies specifically reacting with:

-   (i) a peptide having the 5-10 or 4-11 amino acid sequence in SEQ ID    NO: 1;-   (ii) a peptide having the 6-11 or 5-12 amino acid sequence in SEQ ID    NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6;-   (iii) a peptide having the 8-13 or 7-14 amino acid sequence in SEQ    ID NO: 5;-   (iv) a peptide having the 2-7 amino acid sequence in SEQ ID NO: 7;-   (v) a peptide having the 8-13 or 7-14 amino acid sequence in SEQ ID    NO: 8; and,-   (vi) peptides wherein a part of amino acid residues (e.g., one amino    acid residue) in these polypeptides is substituted with a    substitutable group, and the like.

More preferably, the antibodies specifically reacting with the partialpeptides in the C-terminal region in the peptide of the presentinvention are monoclonal antibodies. Preferred examples of themonoclonal antibodies are a monoclonal antibody shown by AUII5-6-10acapable of being produced from a hybridoma cell shown by AUII5-6-10(FERM BP-8221), a monoclonal antibody shown by AUII 103-5-41a capable ofbeing produced from a hybridoma cell shown by AUII103-5-41(FERMBP-8220), etc.

As such, the antibodies specifically reacting with the partial peptidein the C-terminal region in the peptide of the present invention arecapable of reacting with the peptide of the present invention byrecognizing a specific amino acid sequence at the C terminus of thepeptide of the present invention.

The antibodies specifically reacting with the partial peptides in theN-terminal region of the peptide of the present invention may be anyantibodies that are capable of specifically reacting with the partialpeptides in the N-terminal region of the peptide of the presentinvention. Examples of such antibodies include antibodies specificallyreacting with (i) a peptide having the 1-5 amino acid sequence in SEQ IDNO: 1, (ii) a peptide having the 1-6 amino acid sequence in SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6, (iii) a peptide havingthe 1-8 amino acid sequence in SEQ ID NO: 5, (iv) a peptide having the1-8 amino acid sequence in SEQ ID NO: 8, and (v) peptides wherein a partof amino acid residues (e.g., one) in these polypeptides is substitutedwith a substitutable group, and the like.

As the antibodies specifically reacting with the partial peptides in theN-terminal region of the peptide of the present invention, monoclonalantibodies are preferred. More preferred examples of such antibodies areantibodies which are capable of specifically reacting with the partialpeptides in the N-terminal region of the peptide of the presentinvention but are not reactive any partial peptide in the C-terminalregion.

As such, the antibodies specifically reacting with the partial peptidesin the N-terminal region of the peptide of the present invention can bereacted with the peptide of the present invention by recognizing aspecific amino acid sequence at the N terminus in the peptide of thepresent invention described above.

Preparation of antigens for the antibodies of the present invention andproduction of the antibodies are described below.

(1) Preparation of Antigen

The antigen used to produce the antibodies of the present inventionincludes, for example, the peptide of the present invention, syntheticpeptides having one or at least two antigenic determinants which are thesame as the antigenic determinant of the peptide of the presentinvention, etc. (which are hereinafter sometimes merely referred to asthe antigen of the present invention).

The peptide of the present invention can be produced (a) from tissues orcells of mammals (e.g., human, bovine, rat, mouse, swine, monkey, etc.),fishes (e.g., goby, etc.) by known methods or modifications, (b) throughchemical synthesis by known peptide synthesis using a peptidesynthesizer, etc., or (c) by culturing a transformant having a DNAencoding the peptide of the present invention.

-   -   (a) Where the antigen of the present invention is prepared from        tissues or cells of those mammals or fish, the antigen of the        present invention can be prepared by homogenizing the tissues or        cells, extracting the homogenate with an acid, an alcohol, etc.,        and applying the combination of salting-out, dialysis, gel        filtration, chromatographies such as reversed phase        chromatography, ion exchange chromatography, affinity        chromatography, etc. to the resulting extract to perform        purification/isolation.    -   (b) Examples of the synthetic peptide used in chemical synthesis        of the antigen of the peptide of the present invention include        peptides having the same structure as in the antigen of the        present invention purified from naturally occurring peptides and        peptides containing 1 or at least 2 amino acid sequences having        the same as the amino acid sequence at an optional portion        consisting of at least 2, preferably at least 3 amino acids in        the amino acid sequence for the peptide of the present        invention, etc.    -   (c) Where the peptide of the present invention is manufactured        using a transformant containing a DNA, the DNA can be prepared        by known cloning methods (those described in, e.g., Molecular        Cloning (2nd ed.; J. Sambrook et al., Cold Spring Harbor Lab.        Press, 1989), etc.). The cloning methods include (1) a method        for obtaining a transformant containing a DNA encoding the        peptide of the present invention by hybridization method from        cDNA library using a DNA probe or DNA primer designed under the        amino acid sequence of the peptide of the present invention, (2)        a method for obtaining a transformant containing a DNA encoding        the peptide of the present invention by PCR method using a DNA        primer designed based on the amino acid sequence of the peptide        of the present invention, and the like.

The peptide of the present invention as an antigen can be prepared (1)by known peptide synthesis method or (2) by cleaving a peptide havingthe amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQID NO: 8 with an appropriate peptidase.

The peptide synthesis may be any of, for example, solid phase synthesismethod and liquid phase synthesis method. That is, the objective peptidecan be produced by condensing the partial peptides or amino acids, whichcan construct the said peptide, with the remaining part of the peptideand, where the product contains protecting groups, removing theseprotecting groups. Known methods for condensation and elimination of theprotecting groups are described in (i) or (ii) below.

-   (i) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience    Publishers, New York (1966)-   (ii) Schroeder and Luebke, The Peptide, Academic Press, New York    (1965)

After completion of the reaction, the peptide can be purified andisolated by a combination of conventional purification methods such assolvent extraction, distillation, column chromatography, liquidchromatography, recrystallization, etc. When the peptide obtained by theabove methods is in a free form, the peptide can be converted into anappropriate salt by a known method; conversely when the peptide isobtained in a salt form, the salt can be converted into a free form by aknown method.

The amide form of the peptide can be prepared using commerciallyavailable resins that are suitable for amide formation. Examples of suchresins include chloromethyl resin, hydroxymethyl resin, benzhydrylamineresin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin,4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin, etc. Usingthese resins, amino acids, in which α-amino groups and functional groupson the side chains are appropriately protected, are condensed on theresin in accordance with the sequence of the objective peptide accordingto known various condensation methods. At the end of the reaction, thepeptide is excised from the resin, at the same time, the respectiveprotecting groups are removed and the objective peptide is obtained.Alternatively, chlorotrityl resin, oxime resin, 4-hydroxybenzoic acidtype resin, etc. are employed to take out the partially protectedpeptide and the protecting groups are removed in a conventional mannerto give the objective peptide.

For condensation of the protected amino acids described above, a varietyof activation reagent available for peptide synthesis can be used, andcarbodiimides are preferably employed. Examples of such carbodiimidesinclude DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide, etc. For activation bythese activation reagents, the protected amino acids in combination witha racemization inhibitor (e.g., HOBt, HOOBt, etc.) are added directly tothe resin, or the protected amino acids are previously activated in theform of symmetric acid anhydrides, HOBt esters or HOOBt esters, followedby adding the thus activated protected amino acids to the resin.Solvents suitable for use to activate the protected amino acids orcondense with the resin may be appropriately chosen from solvents thatare known to be usable for peptide condensation reactions. Examples ofsuch solvents, which are used for the activation of the protected aminoacids or for the condensation with the resin, are acid amides such asN,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.;halogenated hydrocarbons such as methylene chloride, chloroform, etc.;alcohols such as trifluoroethanol, etc.; sulfoxides such asdimethylsulfoxide, etc.; tertiary amines such as pyridine, etc.; etherssuch as dioxane, tetrahydrofuran, etc.; nitriles such as acetonitrile,propionitrile, etc.; esters such as methyl acetate, ethyl acetate, etc.;and appropriate mixtures of these solvents. The reaction temperature isappropriately chosen from the range known to be applicable to peptidebinding reactions and is usually selected in the range of approximately−20° C. to 50° C. The activated amino acid derivatives are usedgenerally in an excess of 1.5 to 4 times. When the condensation is foundto be insufficient for the peptide bond-forming reaction as a result oftests using the ninhydrin reaction, the condensation can be completed byrepeating the condensation reaction without removal of the protectinggroups. When the condensation is yet insufficient even after repeatingthe reaction, unreacted amino acids are acetylated with acetic anhydrideor acetylimidazole to avoid any possible effect on the subsequentreaction.

Examples of the protecting groups used to protect the amino groups ofthe starting compounds include Z, Boc, t-pentyloxycarbonyl,isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z,adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl,2-nitrophenylsulphenyl, diphenylphosphinothioyl, Fmoc, etc. Examples ofthe protecting groups of a carboxyl group include, in addition to a C₁₋₆alkyl group, a C₃₋₈ cycloalkyl group and a C₇₋₁₄ aralkyl group,2-adamantyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacylgroup and benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide,trityl hydrazide and the like.

The hydroxyl group of serine and threonine can be protected through, forexample, its esterification or etherification. Examples of the groupsappropriately used for the esterification include a lower (C₁₋₆)alkanoyl group, such as acetyl group, etc.; an aroyl group such asbenzoyl group, etc., and a group derived from carbonic acid such asbenzyloxycarbonyl group, ethoxycarbonyl group, etc. Examples of a groupsuitable for the etherification include benzyl group, tetrahydropyranylgroup, t-butyl group, etc.

Examples of groups for protecting the phenolic hydroxyl group oftyrosine include Bzl, Cl-Bzl, 2-nitrobenzyl, Br-Z, t-butyl, etc.

Examples of groups used to protect the imidazole moiety of histidineinclude Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, Bom, Bum,Boc, Trt, Fmoc, etc.

Examples of the activated carboxyl groups in the starting materialinclude the corresponding acid anhydrides, azides, activated esters[esters with alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccimide, N-hydroxyphthalimide, HOBt)]. As the activated aminoacids, in which the amino groups are activated in the starting material,the corresponding phosphoric amides are employed.

As a method for eliminating (split off) the protecting groups, forexample, catalytic reduction under hydrogen gas flow in the presence ofa catalyst such as Pd-black, Pd-carbon, etc.; an acid treatment withanhydrous hydrofluoric acid, methanesulfonic acid,trifluoromethanesulfonic acid or trifluoroacetic acid, or a mixturesolution of these acids; a treatment with a base such asdiisopropylethylamine, triethylamine, piperidine, piperazine, etc.; andreduction with sodium in liquid ammonia; or the like. The elimination ofthe protecting groups by the acid treatment described above is carriedout generally at a temperature of approximately −20° C. to 40° C. In theacid treatment, it is efficient to add a cation scavenger such asanisole, phenol, thioanisole, m-cresol, p-cresol, dimethylsulfide,1,4-butanedithiol, 1,2-ethanedithiol, etc. Furthermore,2,4-dinitrophenyl group used as the protecting group for the imidazoleof histidine is removed by a treatment with thiophenol. Formyl groupused as the protecting group of the indole of tryptophan is eliminatedby the aforesaid acid treatment in the presence of 1,2-ethanedithiol,1,4-butanedithiol, etc. as well as by a treatment with an alkali such asa dilute sodium hydroxide solution, dilute ammonia, etc.

Protection of the functional groups that should not be involved in thereaction of the starting materials, protecting groups, elimination ofthe protecting groups and activation of the functional groups involvedin the reaction may be appropriately selected from known groups andknown means.

In another method for obtaining the amides of the peptide, for example,the α-carboxyl group of the carboxy terminal amino acid is firstamidated; the peptide chain is then extended to a desired length towardthe amino group side. Thereafter, a peptide in which only the protectinggroup of the N-terminal α-amino group in the peptide chain has beeneliminated from the peptide and a peptide (or amino acids) in which onlythe protecting group of the C-terminal carboxyl group has beeneliminated are prepared. The two peptides are condensed in a mixture ofthe solvents described above. The details of the condensation reactionare the same as described above. After the protected peptide obtained bythe condensation is purified, all the protecting groups are eliminatedby the method described above to be able to give the desired crudepeptide. This crude peptide is purified by various known purificationmeans. Lyophilization of the major fraction is able to give the amide ofthe desired peptide.

To prepare the esterified peptide, for example, the α-carboxyl group ofthe carboxy terminal amino acid is condensed with a desired alcohol toprepare the amino acid ester, which is followed by procedure similar tothe preparation of the amidated peptide above to be able to give theester form of the desired peptide.

The antigen of the present invention may be provided for directimmunization in its immobilized form. The antigen of the presentinvention may also be bound or adsorbed to an appropriate carrier andthe complex produced may be provided for immunization. A mixing ratio ofthe carrier to the antigen of the present invention (hapten) may be inany ratio of any type, as long as the antibody can be efficientlyproduced to the antigen of the present invention. A high molecularcarrier conventionally used to produce an antibody to a hapten may beused in a weight ratio of 0.1 to 100 based on 1 of hapten. As such ahigh molecular carrier, there are used a naturally occurring highmolecular carrier and a synthetic high molecular carrier. Examples ofthe naturally occurring high molecular carrier used are serum albuminfrom mammals such as bovine, rabbit, human, etc., thyroglobulins frommammals such as bovine, rabbit, etc., hemoglobins from mammals such asbovine, rabbit, human, sheep, etc or KHL hemocyanin.

As the synthetic high molecular carrier, there may be used, for example,a variety of latexes including polymers or copolymers, etc., such aspolyamino acids, polystyrenes, polyacryls, polyvinyls, polypropylenes,etc.

For coupling of the hapten and the carrier, a variety of condensingagents can be used. Examples of the condensing agents, which areadvantageously employed, are diazonium compounds such as bis-diazotizedbenzidine through crosslinking of tyrosine, histidine or tryptophan;dialdehyde compounds such as glutaraldehyde, etc. through crosslinkingof amino groups therebetween; diisocyanate compounds such astoluene-2,4-diisocyanate, etc.; dimaleimide compounds such asN,N′-o-phenylenedimaleimide, etc. by crosslinking of thiolstherebetween; maleimide activated ester compounds by crosslinking of anamino group with a thiol group; carbodiimide compounds by crosslinkingof an amino group with a carboxyl group; etc. In the crosslinking ofamino groups with each other, one amino group is reacted with anactivated ester reagent (e.g., SPDP, etc.) having dithiopyridyl and thenreduced to introduce the thiol group, whereas another amino group isintroduced with a maleimide group using a maleimide activated esterreagent, and the two groups may be reacted with each other.

(2) Preparation of Monoclonal Antibody

The antigen of the present invention is administered to warm-bloodedanimal independently itself or together with carriers or diluents to thesite where the production of antibody is possible by administrationroutes such as intraperitoneally, intravenously, subcutaneously, etc. Inorder to potentiate the antibody productivity upon the administration,complete Freund's adjuvants or incomplete Freund's adjuvants may beadministered. The administration is usually carried out once in every 2to 6 weeks and approximately 2 to 10 times in total. Examples of thewarm-blooded animal are monkeys, rabbits, dogs, guinea pigs, mice, rats,sheep, goats, chicken, etc. with mice being preferred for thepreparation of monoclonal antibodies.

In the preparation of monoclonal antibodies, from warm-blooded animals,e.g., mice, immunized with the antigen of the present invention, theanimal wherein the antibody titer is noted is selected, then the spleenor lymph node is collected after 2 to 5 days from the final immunizationand antibody-producing cells contained therein are fused with myelomacells to give hybridomas capable of producing monoclonal antibodies tothe peptide of the present invention. Measurement of the antibody titerof the peptide of the present invention in antisera may be made, forexample, by reacting a labeled form of the peptide of the presentinvention, which will be described later, with the antiserum followed byassaying the binding activity of a marker bound to the antibody. Thefusion may be operated, for example, by the known Kohler and Milsteinmethod [Nature, 256, 495 (1975)]. Examples of fusion accelerators arepolyethylene glycol (PEG), Sendai virus, etc., of which PEG ispreferably employed. Examples of the myeloma cells are NS-1, P3U1,SP2/0, AP-1, etc. In particular, P3U1 or the like is preferablyemployed. A preferred ratio in count of the antibody-producing cells(spleen cells) to the myeloma cells used is within a range ofapproximately 1:1 to 20:1. When PEG (preferably, PEG 1000 to PEG 6000)is added in a concentration of approximately 10 to 80% followed byincubation generally at 20 to 40° C., preferably at 30 to 37° C.generally for 1 to 10 minutes, an efficient cell fusion can be carriedout.

Various methods can be used for screening hybridomas capable ofproducing the antibodies of the present invention. Examples of suchmethods include a method which comprises adding the hybridomasupernatant to a solid phase (e.g., microplate) adsorbed with thepeptide of the present invention or its partial peptides directly ortogether with a carrier, then adding an anti-immunoglobulin antibody(when mouse cells are used for the cell fusion, anti-mouseimmunoglobulin antibody is used) labeled with a radioactive substance,an enzyme or the like, or Protein A and detecting the monoclonalantibodies of the present invention bound to the solid phase; a methodwhich comprises adding the hybridoma supernatant to a solid phaseadsorbed with an anti-immunoglobulin antibody or Protein A, adding thepeptide of the present invention labeled with a radioactive substance,an enzyme, etc. and detecting the monoclonal antibodies of the presentinvention bound to the solid phase; etc. Screening and plating of themonoclonal antibodies of the present invention can be performedgenerally in a medium for animal cells (e.g., RPMI 1640) containing10-20% fetal calf serum and supplemented with HAT (hypoxanthine,aminopterin and thymidine). The antibody titer in the hybridomas culturesupernatant can be assayed as in the assay for the antibody titer of theantibody of the present invention in the antisera described above.

Separation and purification of the monoclonal antibody to the peptide ofthe present invention can be carried out by methods applied toconventional separation and purification of immunoglobulins, as in theconventional methods for separation and purification of polyclonalantibodies (e.g., salting-out, alcohol precipitation, isoelectric pointprecipitation, electrophoresis, adsorption and desorption with ionexchangers (e.g., DEAE), ultracentrifugation, gel filtration, or aspecific purification method which involves collecting only an antibodywith an activated adsorbent such as an antigen-binding solid phase,Protein A, Protein G, etc. and dissociating the binding to obtain theantibody; and the like).

As described above, the antibody of the present invention can beproduced by culturing hybridoma cells in a warm-blooded animal in vivoor in vitro and collecting the antibody of the present invention fromthe body fluids or culture.

The antibody of the present invention can sensitively quantify thepeptide of the present invention.

Hereinafter, uses of the antibody of the present invention including themethod of quantifying the peptide of the present invention(immunoassay), pharmaceuticals comprising the antibody of the presentinvention, etc. are described in detail.

(1) Method of Quantifying the Peptide of the Present Invention

Using the antibody of the present invention, the peptide of the presentinvention can be assayed and also detected by tissue staining, or thelike. For these purposes, the antibody molecule itself may be used, orF(ab′)2, Fab′ or Fab fractions of the antibody molecule may be used.

The quantification method using the antibody of the present invention isnot particularly limited. Any quantification method can be used, so longas the amount of antibody, antigen or antibody-antigen complexcorresponding to the amount of antigen (e.g., the amount of the peptideof the present invention) in a fluid to be tested can be detected bychemical or physical means and the amount of the antigen can becalculated from a standard curve prepared from standard solutionscontaining known amounts of the antigen.

For such an assay method, for example, the sandwich method, thecompetitive method, the immunometric method, nephrometry, etc. are used,and the competitive method described below are more preferred in termsof sensitivity and specificity.

1) Competitive Method

The competitive method is the determination method for quantifying thepeptide of the present invention in a test fluid by competitivelyreacting the antibody of the present invention, the test fluid and alabeled form of the peptide of the present invention, and measuring aratio of the labeled form of the peptide of the present invention boundto the antibody.

Preferably, quantification of the peptide of the present invention in atest fluid by the competitive method is carried out using, e.g., solidphase technique.

Specifically, there are the following procedures using anti-mouse IgGantibody as an antibody for solid phase, which comprise:

-   (a) reacting (i) the antibody of the present invention (e.g., the    monoclonal antibody shown by AUII5-6-10a or AUII103-5-41a,    etc.), (ii) the peptide of the present invention, which is labeled    with biotin and (iii) a test fluid; adding avidin labeled with HRP    (horse radish peroxidase) to the plate; after the reaction, assaying    the HRP activity adsorbed onto the solid phase to quantify the    peptide of the present invention;-   (b) adding (i) the antibody of the present invention (e.g., the    monoclonal antibody shown by AUII5-6-10a or AUII103-5-41a,    etc.), (ii) the peptide of the present invention, which is labeled    with HRP and (iii) a test fluid; after the reaction, assaying the    HRP activity adsorbed onto the solid phase to quantify the peptide    of the present invention; etc.    2) Sandwich Method

The sandwich method is a determination method for quantify the peptideof the present invention in a test fluid by reacting the antibody of thepresent invention immobilized on a carrier with a labeled form of theantibody of the present invention and a test fluid, and assaying theactivity of a marker to quantify the peptide of the present invention inthe test fluid.

Preferably, the sandwich method includes:

-   (i) A determination method of the peptide of the present invention    in a test fluid, which comprises reacting the antibody specifically    reacting with a partial peptide in the N-terminal region of the    peptide of the present invention immobilized onto a carrier, a    labeled form of the antibody (monoclonal antibody shown by    AUII5-6-10a or AUII103-5-41a) specifically reacting with a partial    peptide in the C-terminal region of the peptide of the present    invention and the test fluid, and assaying the activity of a    labeling agent;-   (ii) A determination method of the peptide of the present invention    in a test fluid, which comprises reacting the antibody (monoclonal    antibody shown by AUII5-6-10a or AUII103-5-41a) specifically    reacting with a partial peptide in the C-terminal region of the    peptide of the present invention immobilized onto a carrier, a    labeled form of the antibody specifically reacting with a partial    peptide in the N-terminal region of the peptide of the present    invention and the test fluid, and assaying the activity of a    labeling agent; etc.

In the sandwich method, a test fluid is reacted with the immobilizedantibody specifically reacting with a partial peptide in the C-terminalregion of the peptide of the present invention, or the antibodyspecifically reacting with a partial peptide in the N-terminal region ofthe peptide of the present invention (primary reaction) and then thetest fluid is reacted with a labeled antibody specifically reacting witha partial peptide in the C-terminal region of the peptide of the presentinvention, or a labeled antibody specifically reacting with a partialpeptide in the N-terminal region of the peptide of the present invention(secondary reaction), and the activity of a labeling agent on theimmobilizing carrier is assayed, whereby the amount of the peptide ofthe present invention in the test fluid can be quantified. The primaryand secondary reactions may be performed simultaneously or at timeintervals. The labeling agent and immobilizing methods may be based onthose described above. Further, in immunoassay by the sandwich method,the antibodies used for solid phase or for labeling are not necessarilyone species, but a mixture of two or more species of antibodies may beused for purposes of increasing the measurement sensitivity, etc. In themethod of assaying the peptide of the present invention by the sandwichmethod, for example, when the antibodies used in the primary reactionrecognize the partial peptides in the C-terminal region of the peptideof the present invention, the antibodies used in the secondary reactionare preferably those recognizing partial peptides other than theC-terminal region (i.e., the N-terminal region). When the antibodiesused for the primary reaction recognize partial peptides in theN-terminal region of the peptide of the present invention, theantibodies used in the secondary reaction, antibodies recognizingpartial peptides other than the N-terminal region (i.e., the C-terminalregion) are preferably employed.

3) Immunometric Method

In immunometric method, an antigen in a test fluid and an antigenimmobilized to a solid phase are competitively reacted with a givenamount of a labeled antibody of the present invention, followed byseparating the solid phase from the liquid phase; or the antigen in atest fluid is reacted with an excess amount of a labeled antibody of thepresent invention, then an antigen immobilized to a solid phase is addedto bind a unreacted, labeled antibody of the present invention to thesolid phase, followed by separating the solid phase from the liquidphase. Next, the labeling amount in any of the phases is measured todetermine the amount of the antigen in the test fluid.

4) Nephrometry

In nephrometry, the amount of insoluble sediment, which is produced as aresult of the antigen-antibody reaction in a gel or in a solution, ismeasured. When the amount of an antigen in a test fluid is small andonly a small amount of the sediment is obtained, laser nephrometryutilizing laser scattering can be suitably used.

In the quantification methods 1) through 4) described above, labelingagents used for the assay method using labeling substances are notparticularly limited but radioisotopes, enzymes, fluorescent substances,luminescent substances, etc. are employed. Preferred examples of theradioisotopes include, but are not limited thereto, [¹²⁵I], [¹³¹I],[³H], [¹⁴C], etc. The enzymes described above are not particularlylimited but are preferably enzymes which are stable and have a highspecific activity, and include β-galactosidase, β-glucosidase, analkaline phosphatase, a peroxidase, malate dehydrogenase, etc. Thefluorescent substances described above are not particularly limited butexamples include fluorescamine, fluorescein isothiocyanate, etc. Theluminescent substances described above are not particularly limited butexamples include luminol, a luminol derivative, luciferin, lucigenin,etc. Furthermore, the compounds of the biotin-avidin system may be usedfor binding of an antibody to a labeling agent.

For immobilization of antigen or antibody, physical adsorption may beused. Chemical binding techniques conventionally used forinsolubilization or immobilization of proteins, enzymes, etc. may alsobe used. For carriers, there are used, e.g., insoluble polysaccharidessuch as agarose, dextran, cellulose, etc.; synthetic resin such aspolystyrene, polyacrylamide, silicon, etc., and glass or the like.

In applying each of these immunoassays to the method of the presentinvention, it is not necessary to set any special condition, operation,etc. The assay system for the peptide of the present invention may beconstructed in addition to the conditions or operations conventionallyused for each of the methods, taking into account the technicalconsideration of one skilled in the art. For the details of suchconventional technical means, reference may be made to a variety ofreviews, reference books, etc. (for example, Hiroshi Irie (ed.):“Radioimmunoassay” (published by Kodansha, 1974); Hiroshi Irie (ed.):“Radioimmunoassay; Second Series” (published by Kodansha, 1979); EijiIshikawa, et al. (ed.): “Enzyme Immunoassay” (published by Igaku Shoin,1978); Eiji Ishikawa, et al. (ed.): “Enzyme Immunoassay” (SecondEdition) (published by Igaku Shoin, 1982); Eiji Ishikawa, et al. (ed.):“Enzyme Immunioassay” (Third Edition) (published by Igaku Shoin, 1987);“METHODS IN ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A));ibid., Vol. 73 (Immunochemical Techniques (Part B)); ibid., Vol. 74(Immunochemical Techniques (Part C)); ibid., Vol. 84 (ImmunochemicalTechniques (Part D: Selected Immunoassays)); ibid., Vol. 92(Immunochemical Techniques (Part E: Monoclonal Antibodies and GeneralImmunoassay Methods)); ibid., Vol. 121 (Immunochemical Techniques (PartI: Hybridoma Technology and Monoclonal Antibodies)) (all published byAcademic Press); etc.). Thus, the antibody of the present inventionenables to quantify the peptide of the present invention with highsensitivity and is useful for clarification of the physiologicalfunctions of the peptide of the present invention and for theprevention/treatment or diagnosis of diseases/symptoms associated withthe peptide of the present invention.

The peptide of the present invention has effects including a vascularsmooth muscle contractile effect, a myocardiotrophic action, an anxietyincreasing action, etc.

By determining the amount of the peptide of the present inventioncontained in body fluids (blood, plasma, serum, urine, etc.) using theantibody of the present invention, it is possible to diagnose fordiseases associated with the peptide of the present invention [forexample, central nerve diseases (e.g., Alzheimer's disease, Parkinsoniansyndrome, Pick's disease, Huntington's disease, senile dementia,cerebrovascular dementia, etc.), mental disorders (e.g., anxiety,depression, insomnia, schizophrenia, phobia, etc.), circulatory diseases(e.g., hypertension, hypotension, etc.), heart diseases (e.g., heartfailure, arrhythmia, long QT syndrome, dilated congestivecardiomyopathy, hypertrophic cardiomyopathy, pulmonary hypertension,etc.), renal diseases (e.g., nephritis, renal failure, interstitialrenal disorders, etc.), urinary tract disorders (e.g., pollakiuria,urinary incontinence, etc.), or the like] and so on. In addition, theantibody of the present invention can be used to detect the peptide ofthe present invention in test fluids such as body fluids, tissues, etc.Moreover, the antibody of the present invention is available forpreparation of antibody columns used to purify the peptide of thepresent invention, detection of the peptide of the present invention ineach fraction upon purification, an analysis of the behavior of thepeptide of the present invention in cells to be tested; etc.

-   (2) Pharmaceutical Comprising the Antibody of the Present Invention

The antibody of the present invention has the effects of neutralizingthe peptide of the present invention to inhibit the effects exhibited bythe peptide of the present invention, such as the vascular smooth musclecontractile effect, myocardiotrophic action, anxiety increasing action,etc. Thus, the antibody of the present invention can be used aspharmaceuticals such as preventive/therapeutic agents or diagnosticagents, etc. for the diseases associated with the peptide of the presentinvention [for example, central nerve diseases (e.g., Alzheimer'sdisease, Parkinsonian syndrome, Pick's disease, Huntington's disease,senile dementia, cerebrovascular dementia, etc.), mental disorders(e.g., anxiety, depression, insomnia, schizophrenia, phobia, etc.),circulatory diseases (e.g., hypertension, hypotension, etc.), heartdiseases (e.g., heart failure, arrhythmia, long QT syndrome, dilatedcongestive cardiomyopathy, hypertrophic cardiomyopathy, pulmonaryhypertension, etc.), renal diseases (e.g., nephritis, renal failure,interstitial renal disorders, etc.), urinary tract disorders (e.g.,pollakiuria, urinary incontinence, etc.), or the like].

The preventive/therapeutic agent comprising the antibody of the presentinvention is safe and low toxic, and can be administered parenterally ororally to human or mammals (e.g., rats, rabbits, sheep, swine, bovine,cats, dogs, monkeys, etc.) as a liquid preparations or as apharmaceutical composition of appropriate dosage form.

The antibody of the present invention may be administered in its intactform or in the form of an appropriate pharmaceutical composition. Thepharmaceutical composition used for administration may contain theantibody of the present invention or its salt, a pharmacologicallyacceptable carrier and a diluent or an excipient. Such a pharmaceuticalcomposition is provided in a dosage form suitable for oral or parenteraladministration.

Examples of the composition for parenteral administration are injectablepreparations, suppositories, etc. The injectable preparations mayinclude dosage forms such as intravenous, subcutaneous, intracutaneousand intramuscular injections, drip infusions, etc. These injectablepreparations may be prepared by known methods. For example, theinjectable preparations may be prepared by dissolving, suspending oremulsifying the antibody of the present invention or its salt describedabove in a sterile aqueous medium or an oily medium conventionally usedfor injections. As the aqueous medium for injections, there are, forexample, physiological saline, an isotonic solution containing glucoseand other auxiliary agents, etc., which may be used in combination withan appropriate dissolution aid such as an alcohol (e.g., ethanol), apolyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionicsurfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mols)adduct of hydrogenated castor oil)), etc. As the oily medium, there areemployed, e.g., sesame oil, soybean oil, etc., which may be used incombination with a dissolution aid such as benzyl benzoate, benzylalcohol, etc. The prepared injection is preferably filled in anappropriate ampoule. The suppository used for rectal administration maybe prepared by blending the aforesaid antibody or its salt withconventional bases for suppositories.

The composition for oral administration includes a dosage form of solidor liquid, more specifically, tablets (including dragees and film-coatedtablets), pills, granules, powders, capsules (including soft capsules),syrups, emulsions, suspensions, etc. Such a composition is manufacturedby known methods and may contain carriers, diluents or excipientsconventionally used in the field of pharmaceutical preparations. As thecarriers and excipients for tablets e.g., lactose, starch, sucrose andmagnesium stearate are used.

Advantageously, the pharmaceutical compositions for parenteral or oraluse described above are prepared into pharmaceutical preparations with aunit dose suited to fit a dose of the active ingredients. Such unit dosepreparations include, for example, tablets, pills, capsules, injections(ampoules) and suppositories. The amount of the antibody contained isgenerally about 5 to about 500 mg per dosage unit form; it is preferredthat the aforesaid antibody is contained in about 5 to about 100 mgespecially in the form of injection, and in about 10 to 250 mg for theother forms.

Each of the compositions described above may further contain otheractive ingredients, unless any adverse interaction occurs due toblending with the antibody described above.

The dose of the preventive/therapeutic agent or diagnostic agent(pharmaceutical) comprising the antibody of the present invention mayvary depending on subject to be administered, diseases to beadministered, symptoms, routes for administration, etc. When used forthe treatment of, e.g., obesity in an adult patient, it is advantageousthat the antibody of the present invention is intravenously administeredin a single dose of normally approximately 0.01 to 20 mg/kg body weight,preferably approximately 0.1 to 10 mg/kg body weight and more preferablyapproximately 0.1 to 5 mg/kg body weight approximately 1 to 5 times,preferably approximately 1 to 3 times a day. For other parenteraladministrations (e.g., subcutaneous administration) and oraladministration, the corresponding dose may be administered. Whensymptoms are extremely serious, the dose may be increased depending onthe conditions.

In the specification of the present invention, amino acids, etc. areshown by abbreviations and in this case, they are denoted in accordancewith the IUPAC-IUB Commission on Biochemical Nomenclature or by thecommon codes in the art, examples of which are shown below. For aminoacids that may have the optical isomer, L form is presented unlessotherwise indicated.

-   PAM: phenylacetamidomethyl-   Boc: t-butyloxycarbonyl-   Fmoc: 9-fluorenylmethyloxycarbonyl-   Cl-Z: 2-chloro-benzyloxycarbonyl-   Br-Z: 2-bromo-benzyloxycarbonyl-   Bzl: benzyl-   Cl-Bzl: 2-chloro-benzyl-   OcHex: cyclohexyl ester-   OBzl: benzyl ester-   Tos: p-toluenesulfonyl-   HONB: N-hydroxy-5-norbornene-2,3-dicarboximido-   HOBt: 1-hydroxybenzotriazole-   HOOBt: 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine-   MeBzl: 4-methylbenzyl-   Bom: benzyloxymethyl-   Bum: t-butoxymethyl-   Trt: trityl-   DNP: dinitrophenyl-   TFA: trifluoroacetic acid-   DMF: N,N-dimethylformamide-   DCM: dichloromethane-   DCC: N,N′-dichlorohexylcarbodiimide-   BHA: benzhydrylamine-   pMBHA: p-methylbenzhydrylamine-   CHO: formyl-   GlyL glycine-   Ala: alanine-   Val: valine-   Leu: leucine-   Ile: isoleucine-   Ser: serine-   Thr: threonine-   Cys: cysteine-   Met: methionine-   Glu: glutamic acid-   Asp: aspartic acid-   Lys: lysine-   Arg: arginine-   His: histidine-   Phe: phenylalanine-   Tyr: tyrosine-   Trp: tryptophan-   Pro: proline-   Asn: asparagine-   Gln: glutamine

The sequence identification numbers used in the sequence listing of thespecification represents the amino acid sequences of the followingpeptides.

-   [SEQ ID NO: 1]    -   This shows the amino acid sequence of human urotensin II.-   [SEQ ID NO: 2]    -   This shows the amino acid sequence of porcine urotensin II-1.-   [SEQ ID NO: 3]    -   This shows the amino acid sequence of porcine urotensin II-2.-   [SEQ ID NO: 4]    -   This shows the amino acid sequence of bovine urotensin II.-   [SEQ ID NO: 5]

This shows the amino acid sequence of rat urotensin II.

[SEQ ID NO: 6]

This shows the amino acid sequence of goby urotensin II.

[SEQ ID NO: 7]

This shows the amino acid sequence of human urotensin II-related peptide(URP) obtained in REFERENCE EXAMPLE 1 described below.

-   [SEQ ID NO: 8]    -   This shows the amino acid sequence of mouse urotensin II.-   [SEQ ID NO: 9]    -   This shows the amino acid sequence of the sequence of 5-10 amino        acid in (the 6-11 amino acid sequence in SEQ ID NO: 2, SEQ ID        NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6; the 8-13 amino acid        sequence in SEQ ID NO: 5; the 2-7 amino acid sequence in SEQ ID        NO: 7; or the 8-13 amino acid sequence in SEQ ID NO: 8).

The hybridoma cell AUII5-6-10 obtained in EXAMPLE 1 later described hasbeen deposited on International Patent Organisms Depository, NationalInstitute of Advanced Industrial Science and Technology, located atCentral 6, 1-1-1 Higashi, Tsukuba, Ibaraki (postal code: 305-8566) underAccession Number FERM BP-8221 since Oct. 22, 2002.

The hybridoma cell AUII103-5-41 obtained in EXAMPLE 1 later describedhas been deposited on International Patent Organisms Depository,National Institute of Advanced Industrial Science and Technology,located at Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki (postal code:305-8566) under Accession Number FERM BP-8220 since Oct. 22, 2002.

The antibody obtained from each of the hybridoma cells is shown by thecell name followed by “a.”

Hereinafter, the present invention will be described in detail withreference to REFERENCE EXAMPLE and EXAMPLES, but is not deemed to limitthe scope of the present invention.

REFERENCE EXAMPLE 1

Production of human urotensin II-related peptide (URP) (SEQ ID NO: 7)

In a reactor of peptide synthesizer ACT-90 (Advanced ChemTech, Inc.),0.5 mmole (0.77 mmole/g resin) of Boc-Val-OCH₂—PAM resin commerciallyavailable was charged and Boc-Cys (MeBzl), Boc-Tyr(Br-Z), Boc-Lys(Cl-Z),Boc-Trp (CHO), Boc-Phe, Boc-Cys (MeBzl) and Boc-Ala in this order wereintroduced therein in accordance with the Boc-strategy (NMP-HOBt)peptide synthesis to give the objective protected peptide resin. After0.32 g of this resin was distilled with 2 ml of p-cresol and 1.5 ml of1,4-butanedithiol at 0° C. for 60 minutes in 20 ml of anhydrous hydrogenfluoride, hydrogen fluoride was removed in vacuum. Diethyl ether wasadded to the residue and the precipitates were taken out by filtration.To the precipitates 50% aqueous acetic acid solution was added forextraction to remove insoluble matters. After the extract wassufficiently concentrated, the concentrate was applied to a Sephadex(registered trademark) G-25 column (2.0×80 cm) packed with 50% aqueousacetic acid solution, followed by developing with the same solvent tocollect the main fractions. The fractions were lyophilized to give 118mg of crude SH peptide. From the peptide 50 mg was taken and dissolvedin 100 ml of 6M aqueous urea solution. After 400 ml of distilled waterwas added to dilute, pH of the dilution was adjusted to 8 with ammoniawater and the mixture was gently agitated while bubbling air. Thereaction was monitored on HPLC and after it was confirmed on the peaksthat all the SH-form peptides were converted into the SS-form peptides,acetic acid was added to the solution to adjust the pH to 3. Thesolution was applied to a reversed phase chromatography column (2.6×60cm) packed with LiChroprep (registered trademark) RP-18 and washed with200 ml of 0.1% aqueous TFA and then with 200 ml of 20%acetonitrile/water containing 0.1% TFA. Next, linear gradient elutionwas conducted using 300 ml of 20% acetonitrile/water containing 0.1% TFAand 300 ml of a 50% aqueous acetonitrile containing 0.1% TFA. The mainfractions were collected and lyophilized to give 7.9 mg of whitepowders.

-   ESI-MS:M⁺ 1017.1 (calc. 1017.2)-   Elution time on HPLC: 9.9 mins.-   Column conditions-   Column: Wakosil-II 5C18HG 4.6×100 mm-   Eluent: Liner density gradient elution using Eluent A: 0.1%    TFA-water and Eluent B: acetonitrile containing 0.1% TFA in A/B:    80/20˜60/40 (10 mins.)-   Flow rate: 1.0 ml/min.

EXAMPLE 1

(1) Preparation of Immunogens and Immunization

Using as an antigen goby (goby, long-jawed mudsucker, (Gillichthysmirabilis) urotensin II (purchased from Peninsula Laboratories, Inc.,SEQ ID NO: 6) with the C-terminal structure (Cys-Phe-Trp-Lys-Tyr-Cys)identical with human urotensin II (SEQ ID NO: 1), porcine urotensinII-1(SEQ ID NO: 2), porcine urotensin II-2 (SEQ ID NO: 3), bovineurotensin II (SEQ ID NO: 4) and rat urotensin II (SEQ ID NO: 5),antibodies recognizing the C terminus of urotensin II were prepared.

For preparation of the antigen, 1 mg of goby urotensin II peptide wasbound to 4 mg of bovine thyroglobulin (BTG) using 30 mg of ECDI(1-ethyl-3′-(3-dimethylaminopropyl)carbodiimide, Dojin Kagaku). Then,the reaction solution containing the resulting goby urotensin II-BTGcomplex was dialyzed to 0.15 M sodium chloride aqueous solution. Theinternal dialysate was mixed with Freund's complete adjuvant. Using themixture as an antigen, goby urotensin II was applied to Balb/C mice(female, 6-8 weeks old) in an amount of 20 μg/animal for primaryimmunization. Approximately 4 weeks after the primary immunization, thecomplex was mixed with Freund's incomplete adjuvant and the mixture wasused as an antigen for secondary immunization. The animal was boosteredwith a mixture of goby urotensin II-BTG complex and Freund's incompleteadjuvant every 2 other weeks until the antibody titer increased.

(2) Preparation of Biotinylated Antigen

Biotin was bound to goby urotensin II to use as a labeled antigen forenzyme-linked immunoassay (EIA). That is, 2 nmols of goby urotensin IIwas dissolved in 0.1 ml of 50 mM phosphate buffer (pH 7.5) and 20 nmolsof biotin N-hydroxysuccinimide ester was added to the solution. Themixture was reacted at room temperature for an hour. By this reaction,goby urotensin II biotinylated at the α-amino group of N-terminalalanine or goby urotensin II biotinylated at the ε-amino group of 9thlysine and goby urotensin II biotinylated at both groups were produced.The biotinylated products were fractionated on HPLC to obtain gobyurotensin II biotinylated only at the N-terminal alanine residue([N-biotinyl-Ala¹] goby urotensin II, hereinafter referred to asbiotinylated goby urotensin II). The structure of goby urotensin IIbiotinylated only at the N-terminal alanine residue was identified bymass spectrometry to detect an increased molecular weight correspondingto one molecule of biotin bound and by N-terminal amino acid sequencingusing Edman degradation, where the α-amino group of N-terminal alanineresidue was biotinylated so that the reaction with phenyl isothiocyanatedid not proceed and any phenylthiohydantoin derivative of the alanineresidue was not detected at all.

(3) Measurement of Antibody Titers

Antibody titers in mouse anti-sera during immunization of goby urotensinII were measured by the following procedures. First, in order to preparean anti-mouse immunoglobulin antibody-bound microplate, 100 μl each of50 mM carbonate buffer (pH 9.6) containing 10 μg/ml of anti-mouseimmunoglobulin antibody (IgG fraction, manufactured by Cappel Inc.) wasdispensed in a 96-well microplate, which was allowed to stand at 4° C.for 24 hours. Next, the plate was washed with phosphate buffered saline(PBS, pH 7.4). Then, 200 μl each of PBS containing 25% Block Ace(manufactured by Snow Brand Milk Products Co., Ltd.) was dispensed toblock the surplus binding sites, followed by treating the plate at 4° C.for at least 24 hours.

After 100 μl each of mouse anti-goby urotensin II antisera diluted withBuffer C (0.02 M phosphate buffer containing 1% BSA, 0.4 M NaCl and 2 mMEDTA, pH 7.0) was added to each well of the anti-mouse immunoglobulinantibody-bound microplate described above, the mixture was reacted at 4°C. for 16 hours. The plate was then washed with PBS and 100 μl of thebiotinylated goby urotensin II (diluted with Buffer C to 200-fold)prepared in (2) above was added thereto and the mixture was reacted atroom temperature for 6 hours. Next, the plate was washed with PBS and100 μl of HRP (horse radish peroxidase)-labeled avidin solution dilutedwith Buffer C to 10000-fold was then added to each well. The mixture wasreacted at room temperature for 2 hours. After the plate was washed withPBS, 100 μl of TMB microwell peroxidase substrate system (KIRKEGAARD &PERRY LAB, INC., Funakoshi Pharmaceutical Co., Ltd.) was added theretoand the mixture was reacted at room temperature for 10 minutes therebyto assay the enzyme activity on the solid phase. After 100 μl of 1 Mphosphate was added to discontinue the reaction, absorbance at 450 nmwas measured on a plate reader (BICHROMATIC, manufactured by DainipponPharmaceutical Co., Ltd.).

The results are shown in FIG. 1.

Increased antibody titers to the C terminus of goby urotensin II werenoted in 5 out of 8 immunized mice.

(4) Preparation of Monoclonal Anti-Urotensin II Antibodies

A solution of 200-300 μg of the immunogen in 0.25-0.3 ml of saline wasintravenously injected to mouse showing a relatively high antibody titerfor final immunization. The spleen was withdrawn from mouse 3 to 4 daysafter the final immunization, pressed against and filtered through astainless mesh, and suspended in Eagle's minimum essential medium (MEM)to give a spleen suspension. For cell fusion, BALB/C mouse-derivedmyeloma cells P3-X63.Ag8.U1(P3U1) were used (Current Topics inMicrobiology & Immunology, 81, 1, 1978). Cell fusion was carried out bya modification of the originally reported method (Nature, 256, 495,1975). That is, the splenocytes and P3U1 were washed 3 times withserum-free MEM, respectively, to mix the splenocytes with P3U1 in 5:1 interms of cell counts. The mixture was centrifuged at 800 rpm for 15minutes to precipitate the cells. After the supernatant was thoroughlyremoved, the precipitates were lightly loosened and 0.3 ml of 45%polyethylene glycol (PEG) 6000 (manufactured by Kochleit) was addedthereto. The mixture was settled at 37° C. for 7 minutes in a thermostatfor cell fusion. After completion of the fusion, MEM was added to thecells at a rate of 2 ml/min. to reach 15 ml of MEM in total. The mixturewas then centrifuged at 600 rpm for 15 minutes to remove thesupernatant. The cell deposits were suspended in GIT medium (Wako PureChemical Industries, Ltd.) (GIT-0% FCS) supplemented with 10% fetal calfserum in 2×10⁵ cells/ml, and the suspension was plated onto 120 wells ofa 24-well Multidish (manufactured by Linbro Chemical Co.) in 1 mleach/well. After plating, the cells were incubated at 37° C. in a 5% CO₂incubator. Twenty-four hours after, GIT-0% FCS medium containing HAT(1×10⁻⁴ M hypoxanthine, 4×10⁻⁷ M aminopterin and 1.6×10⁻³ M thymidine)(HAT medium) was added to the wells in 1 ml each/well to initiate HATselection culture. After 1 ml of the old medium was discarded on Days 3,6 and 9 subsequent to the culture initiation, HAT selection culture wascontinued by supplementing 1 ml of HAT medium. Hybridomas were found togrow on Days 9 to 14 after the cell fusion. When the culture mediumturned yellow (about 1×10⁶ cells/ml), the supernatant was collected andthe antibody titers were assayed in accordance with the proceduresdescribed in (3) above. In order to confirm binding specificity of theantibody in the hybridoma supernatant to biotinylated goby urotensin II,it was examined at this point of time if the binding would be inhibitedwith 1 μM porcine urotensin II-1.

As a typical example of screening of mouse-derived hybridoma immunizedwith goby urotensin II, the results obtained using mouse No. 6 (seeFIG. 1) are shown in FIG. 2.

The antibodies in the hybridoma supernatants from No. 5 and No. 103 werefound to specifically bind to urotensin II. Thus, total 2 hybridomasfrom No. 5 and No. 103 were selected.

Next, these hybridomas were cloned by limiting dilution. In cloning,thymocytes from BALB/C mice were added as feeder cells to the wells in5×10⁵ cells/well. After the cloning, 2 clones of No. 5-6-10 and No.103-5-41 were selected as hybridomas showing a higher antibodyproduction level. Hybridomas No. 5-6-10 and No. 103-5-41 were namedAUII5-6-10 and AUII103-5-41, respectively.

After cloning, each hybridoma was intraperitoneally given in a dose of 1to 3×10⁶ cells/animal to mice (BALB/C), to which 0.5 ml of mineral oilhad previously been given intraperitoneally, and the ascites containingthe antibodies were collected 6 to 20 days after.

The monoclonal antibodies were purified from the collected ascitesthrough Protein A column. That is, 6 to 20 ml of the ascites was dilutedwith an equal amount of a binding buffer (1.5 M glycine containing 3.5 MNaCl and 0.05% NaN₃, pH 9.0). The dilution was then provided ontoRecombinant Protein A-Agarose (manufactured by Repligen Corp.), whichhad been previously equilibrated with the binding buffer to elute thespecific antibody with an eluting buffer (0.1M citrate buffer containing0.05% NaN₃, pH 3.0). The eluate was dialyzed to PBS at 4° C. for 2 days,then subjected to bacteria-free filtration through 0.22 μm filter(manufactured by Millipore Inc.) and stored at 4° C. or −80° C. Toidentify the class/subclass of the monoclonal antibody, enzyme-linkedimmunosorbent assay (ELISA) using a purified monoclonal antibody-boundsolid phase was carried out. That is, 100 μl each of 0.1 M carbonatebuffer solution, pH 9.6, containing 2 μg/ml of the antibody wasdispensed to a 96-well microplate, which was allowed to stand at 4° C.for 24 hours. Following the procedures described in (3) above, thesurplus binding sites in the wells were blocked with Block Ace.Thereafter, the class/subclass of the immobilized antibodies wasidentified by ELISA using an isotype typing kit (Mouse-Typer™Sub-Isotyping Kit, manufactured by Biorad Inc.). The classes of theantibodies produced from the two hybridomas (No. 5-6-10 and No.103-5-41) were both found to belong to IgG1.

EXAMPLE 2

Enzyme Immunoassay by Competitive Method

The reaction specificity of the monoclonal antibodies (AUII5-6-10a andAUII103-5-41a) produced by the respective two hybridomas No. 5-6-10 andNo. 103-5-41, which were prepared using goby urotensin II as animmunogen, was examined by the following procedures.

To the anti-mouse immunoglobulin antibody-bound microplate described inEXAMPLE 1 (3) above, 33 μl of a 486-fold dilution of the AUII5-6-10hybridoma culture supernatant diluted with Buffer C (0.02 M phosphatebuffer containing 1% BSA, 0.4 M NaCl and 2 mM EDTA, pH 7.0) or 33 μl ofa 54-fold dilution of the AUII103-5-41 hybridoma culture supernatantdiluted with Buffer C, 33 μl each of human, porcine-1, bovine, rat andgoby urotensin II solutions in various concentrations prepared usingBuffer C, and 33 μl of the biotinylated goby urotensin II (diluted withBuffer C to 8333-fold) were added, and each mixture was reacted at 4° C.for 16 hours. After completion of the reaction, the mixture was washedwith PBS and 100 μl of HRP-labeled avidin solution diluted with Buffer Cto 10000-fold was added to each well, followed by reacting at roomtemperature for 3 hours. After completion of the reaction, the mixturewas washed with PBS and the enzyme activity on the solid phase wasassayed by the method described in EXAMPLE 1 (3) above.

The results of competitive EIA obtained using the culture supernatantsof these hybridomas (AUII5-6-10 and AUII1003-5-41) are shown in FIGS. 3and 4.

As shown in FIG. 3, it was noted that AUII5-6-10a displayed a similarreactivity to any of human, porcine-1, bovine, rat and goby urotensin IIpeptides. It is considered based on these results that AUII5-6-10arecognizes the Cys-Phe-Trp-Lys-Tyr-Cys sequence, which is a partialstructure commonly possessed by these peptides. From the bindinginhibition curve of AUII5-6-10a for human urotensin II, the humanurotensin II level for (B/B₀)=0.5 was found to be 1.2 nM.

On the other hand, as shown in FIG. 4, AUII103-5-41a displayed a higherreactivity with human and goby urotensin II, as compared to thereactivity with porcine-1, bovine and rat urotensin II. The reactivityof AUII103-5-41a with human urotensin II (the antigen level which gives(B/B₀)=0.5: 0.68 nM) was about 0.04 time the reactivity with porcineurotensin II-1(the antigen level which gives (B/B₀)=0.5: 17.4 nM). Fromthis it is considered that AUII103-5-41a will recognize theAsp-Cys-Phe-Trp-Lys-Tyr-Cys sequence or the partial structure commonlypossessed in human and goby urotensin II.

The level of human urotensin II, which gives (B/B₀)=0.5 in these twohybridoma culture supernatants, is in a range of 0.5 to 2.0 nM,indicating that the competitive-EIA using these two hybridoma culturesupernatants is highly sensitive. Thus, about 0.2 nM [(B/B₀)=0.9] humanurotensin II could be detected.

EXAMPLE 3

Neutralizing Actions on the Biological Activities of Human, Porcine-1,Bovine, Rat and Goby Urotensin II with Monoclonal Antibody AUII5-6-10a

The neutralizing actions on the biological activities of human,porcine-1, bovine, rat and goby urotensin II with monoclonal antibodyAUII5-6-10a were determined by the assay system for arachidonatemetabolite releasing activity using rat GPR14 receptor expression CHOcells (the same cells as the rat SENR expression CHO cells described inWO 00/32627).

AUII5-6-10a was diluted to various concentrations (1, 3, 10, 30, 100 and300 nM) and the dilution was incubated with human, porcine-1, bovine,rat and goby urotensin II (10 nM each) at room temperature for an hour.The residual activity was then assayed using rat GPR14 receptorexpression CHO cells.

The arachidonate metabolite releasing activity was assayed as follows.Rat GPR14 receptor expression CHO cells were plated on a 24-well plateat a cell density of 0.5×10⁵ cells/well. After incubation for 24 hours,[³H] arachidonic acid was added to the wells in 0.5 μCi/well.Twenty-four hours after the addition of [³H] arachidonic acid, the cellswere washed with MEM containing 0.1% BSA and a solution mixture of themonoclonal antibody in each concentration described above with human,porcine-1, bovine, rat and goby urotensin II was added thereto in 500μl/well. After incubation at 37° C. for an hour, 400 μl out of 500 μl ofthe reaction solution was added to 4 ml of a scintillator to monitor theamount of [³H] arachidonate metabolite released into the reactionsolution on a scintillation counter.

The results are shown in FIG. 5.

AUII5-6-10a suppressed 100% of the activities of human, porcine-1,bovine, rat and goby urotensin II by 3-fold or 10-fold amount (in amolar ratio).

The foregoing results reveal that AUII5-6-10a neutralizes thearachidonate metabolite releasing activities of human, porcine-1,bovine, rat and goby urotensin II.

EXAMPLE 4

Anti-Anxiety-Like Action of AUII5-6-10a in the Hole-Board Test System

ICR (CD-1) mice (9-19 weeks old weighing 36-38 g, male, Charles RiverJapan, Inc.) were mildly anesthetized with diethyl ether and mouse IgG(immunoglobulin G) (Sigma, 10 mg/ml PBS, 5 μl) or AUII5-6-10a (10 mg/mlPBS, 5 μl) was given to the right ventricle. A double needle (MatsumotoSeisakusho) was used for intraventricular administration. Thirty minutesafter the mice awaken were placed in a restraint cage (NatsumeSeisakusho Co., Ltd.) and exposed to restraint stress for an hour.Control animal was allowed to freely behave in a breeding cage for anhour. Spontaneous locomotor activity and peeping behavior werequantified for subsequent 5 minutes. For the measurement, thespontaneous motor activity monitoring system (Muromachi Kikai Co., Ltd.)was employed. The peeping behavior of mice was quantified with anapparatus consisting of a rearing sensor (MRX-110TX-RX) equipped at thelower side of the cage and a board with holes therethrough (3.8 cm indiameter, 4 holes) being suspended from the upper part by 5 mm above thesensor to fix inside the cage. The number of peeping was expressed interms of the intercepting count of the mounted sensor with mice placedon the board by poking the head out from the holes due to its peepingbehavior. At the same time, total spontaneous motor activity wasmeasured with a SUPERMEX sensor (PYS-001, a passive infrared sensor)mounted to the upper part. The total spontaneous motor activity wasexpressed by the number that mice crossed the sensor. Experiment wascarried out between 15:00 and 18:00.

The results are shown below (FIG. 6).

Administered with mouse IgG (non-stressed): 70.5±4.2 counts (n=21)

Administered with mouse IgG (exposed to restraint stress): 51.1±5.3counts (n=19), p<0.1

Administered with AUII5-6-10a (exposed to restraint stress): 64.6±3.6counts (n=21), p<0.05

These results reveal that by exposure to restraint stress for an hour,the peeping behavior of mice was significantly reduced and further thatintraventricular administration of AUII5-6-10a significantly recoveredthe peeping behavior reduced by the restraint stress.

In this case, no change in the total spontaneous motor activity of micewas observed [administered with mouse IgG (non-stressed): 621.5±20.4counts; administered with mouse IgG (exposed to restraint stress):611.2±29.6 counts; administered with AUII5-6-10a (exposed to restraintstress): 649.7±20.1 counts]

The foregoing results reveal that intraventricular administration ofAUII5-6-10a suppressed anxiety-like behavior shown by the reducedpeeping behavior of mice induced by restraint stress, without affectingany spontaneous behavior.

INDUSTRIAL APPLICABILITY

The antibody of the present invention is useful for development oftherapeutic agents, preventive agents and diagnostic agents for diseasesassociated with the peptide of the present invention. By using hybridomacells containing the antibody of the present invention, the antibody ofthe present invention can be manufactured in an industrial scale. Inaddition, the pharmaceuticals (especially diagnostic agents) comprisingthe antibody of the present invention are useful for diagnosis ofdiseases associated with the peptide of the present invention [forexample, central nerve diseases (e.g., Alzheimer's disease, Parkinsoniansyndrome, Pick's disease, Huntington's disease, senile dementia,cerebrovascular dementia, etc.), mental disorders (e.g., anxiety,depression, insomnia, schizophrenia, phobia, etc.), circulatory diseases(e.g., hypertension, hypotension, etc.), heart diseases (e.g., heartfailure, arrhythmia, long QT syndrome, dilated congestivecardiomyopathy, hypertrophic cardiomyopathy, pulmonary hypertension,etc.), renal diseases (e.g., nephritis, renal failure, interstitialrenal disorders, etc.), urinary tract disorders (e.g., pollakiuria,urinary incontinence, etc.), or the like].

The antibody of the present invention has the activity of neutralizingthe peptide of the present invention and hence, is useful as thepreventive/therapeutic agent for central nerve diseases (e.g.,Alzheimer's disease, Parkinsonian syndrome, Pick's disease, Huntington'sdisease, senile dementia, cerebrovascular dementia, etc.), mentaldisorders (e.g., anxiety, depression, insomnia, schizophrenia, phobia,etc.), circulatory diseases (e.g., hypertension, hypotension, etc.),heart diseases (e.g., heart failure, arrhythmia, long QT syndrome,dilated congestive cardiomyopathy, hypertrophic cardiomyopathy,pulmonary hypertension, etc.), renal diseases (e.g., nephritis, renalfailure, interstitial renal disorders, etc.), urinary tract disorders(e.g., pollakiuria, urinary incontinence, etc.), or the like.

By using the antibody of the present invention, the amount of thepeptide of the present invention can be measured with a highsensitivity. Therefore, the quantifying method of the present inventionis useful for diagnosis, prevention or treatment for the diseasesassociated with the peptide of the present invention [for example,central nerve diseases (e.g., Alzheimer's disease, Parkinsoniansyndrome, Pick's disease, Huntington's disease, senile dementia,cerebrovascular dementia, etc.), mental disorders (e.g., anxiety,depression, insomnia, schizophrenia, phobia, etc.), circulatory diseases(e.g., hypertension, hypotension, etc.), heart diseases (e.g., heartfailure, arrhythmia, long QT syndrome, dilated congestivecardiomyopathy, hypertrophic cardiomyopathy, pulmonary hypertension,etc.), renal diseases (e.g., nephritis, renal failure, interstitialrenal disorders, etc.), urinary tract disorders (e.g., pollakiuria,urinary incontinence, etc.), or the like]. By using the antibody of thepresent invention, the amount of the peptide of the present inventionderived from human, swine, bovine, rat, mouse, goby, etc. can bedetermined with a high sensitivity and is thus useful as the diagnosticagent, preventive/therapeutic agent, reagent, etc. for diseasesassociated with the peptide of the present invention.

1. An antibody reacting specifically reacting with a partial peptide inthe C-terminal region of a polypeptide having the amino acid sequencerepresented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8, or a derivative of saidpolypeptide:
 2. The antibody according to claim 1, which specificallyreacts with a partial peptide in the C-terminal region of a polypeptidehaving the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or aderivative of said polypeptide.
 3. The antibody according to claim 1,which specifically reacts with a peptide having the amino acid sequencerepresented by SEQ ID NO:
 9. 4. The antibody according to claim 1,wherein the partial peptide in the C-terminal region is a peptide having(i) the 5-10 amino acid sequence in SEQ ID NO: 1, (ii) the 6-11 aminoacid sequence in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:6, (iii) the 8-13 amino acid sequence in SEQ ID NO: 5, (iv) the 2-7amino acid sequence in SEQ ID NO: 7, or (v) the 8-13 amino acid sequencein SEQ ID NO:
 8. 5. The antibody according to claim 1, which is amonoclonal antibody.
 6. The antibody according to claim 1, which islabeled.
 7. The antibody according to claim 1, which is a neutralizingantibody.
 8. The antibody according to claim 7, which neutralizes theactivity of a polypeptide having the amino acid sequence represented bySEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7 or SEQ ID NO: 8, or a derivative thereof.
 9. The antibodyaccording to claim 7, which neutralizes the activity of a polypeptidehaving the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:
 6. 10. The antibodyaccording to claim 5, which is shown by AUII5-6-10a capable of beingproduced from a hybridoma cell shown by AUII5-6-10 (FERM BP-8221). 11.The antibody according to claim 5, which is shown by AUII103-5-41acapable of being produced from a hybridoma cell shown by AUII103-5-41(FERM BP-8220).
 12. A hybridoma cell capable of producing the antibodyaccording to claim
 5. 13. The hybridoma cell according to claim 12,which is shown by AUII103-5-41 (FERM BP-8220).
 14. The hybridoma cellaccording to claim 12, which is shown by AUII5-6-10 (FERM BP-8221). 15.A method of producing the antibody according to claim 5, which comprisesculturing the hybridoma cell according to claim 12 in vivo or in vitroand collecting the antibody according to claim 5 from the body fluid orits culture.
 16. A pharmaceutical comprising the antibody according toclaim
 1. 17. The pharmaceutical according to claim 16, which is apreventive/therapeutic agent for central nerve diseases, mentaldisorders, circulatory diseases, heart diseases, renal diseases orurinary tract disorders.
 18. A diagnostic agent comprising the antibodyaccording to claim
 1. 19. The diagnostic agent according to claim 18,which is a diagnostic agent for central nerve diseases, mentaldisorders, circulatory diseases, heart diseases, renal diseases orurinary tract disorders.
 20. A method of quantifying a polypeptidehaving the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:8, or a derivative thereof, which comprises using the antibody accordingto claim
 1. 21. A method of quantifying a polypeptide having the aminoacid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8, or aderivative thereof, in a test fluid, which comprises competitivelyreacting the antibody according to claim 1, a test fluid and a labeledform of polypeptide having the amino acid sequence represented by SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or a derivative of saidpolypeptide, and determining a ratio of the labeled polypeptide havingthe amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQID NO: 8, or a derivative thereof, bound to the antibody.
 22. A methodfor diagnosis of a disease associated with a polypeptide having theamino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ IDNO: 8, or a derivative thereof, which comprises using the antibodyaccording to claim
 1. 23. A method of preventing/treating central nervediseases, mental disorders, circulatory diseases, heart diseases, renaldiseases or urinary tract disorders, which comprises administering aneffective dose of the antibody according to claim 1 to a mammal.
 24. Useof the antibody according to claim 1 for manufacturing apreventive/therapeutic agent for central nerve diseases, mentaldisorders, circulatory diseases, heart diseases, renal diseases orurinary tract disorders.